1. Field of the Invention
The present invention relates to a C3 plant which contains a gene for an enzyme involved in a C4 pathway of photosynthesis (hereinafter, referred to as a C4 photosynthesis gene) and expresses the C4 photosynthesis gene at a high level.
2. Description of the Related Art
Plants are classified into C3 plants, C4 plants, and crassulacean acid metabolism (CAM) plants, based on the kind of initial fixed products in photosynthetic fixation of CO2. Ninety percent or more of plants on the earth belong to C3 plants, which include, for example, agriculturally important plants such as rice and barley. The photosynthetic pathway of C3 plants is also called the Calvin pathway, and an enzyme involved in photosynthetic fixation of CO2 in this pathway is ribulose-1,5-bisphosphate carboxylase. This enzyme has a low affinity for CO2 and has a high affinity for O2. Therefore, the efficiency of a photosynthetic reaction as well as photosynthetic fixation of CO2 is low in the C3 photosynthetic pathway.
The C4 plants are those which have evolved so as to overcome such non-efficient photosynthetic fixation of CO2. The C4 plants have a mechanism for concentrating CO2 and a high photosynthetic capacity. An enzyme involved in photosynthetic fixation of CO2 in the photosynthetic pathway of the C4 plants is phosphoenolpyruvate carboxylase. This enzyme has a high capacity of photosynthetic fixation of CO2 without its activity being inhibited by O2.
CAM plants have a photosynthetic system suitable for dry environment, and the photosynthetic system is considered to be a sort of evolved form of the C3 photosynthetic system.
It is expected that the photosynthetic capacity and productivity of the agriculturally important C3 plants (e.g., rice) will be remarkably improved by providing a C3 plant with the photosynthetic function of a C4 plant. Some attempts have been made to introduce a C4 photosynthesis gene into a C3 plant.
In order to express a photosynthesis gene of a C4 plant, a chlorophyll a/b binding protein promoter or a Cauliflower mosaic virus (CaMV) 35S promoter linked thereto has been used. For example, there is a report by Kogami et al., Transgenic Research 3: 287-296 (1994): The (CaMV) 35S promoter which can be expressed at a high level in a leaf tissue of a C3 plant was linked to a photosynthesis gene of a C4 plant (the phosphoenolpyruvate carboxylase (PEPC) gene) to produce recombinant DNA, and then the recombinant DNA was introduced into a C3 plant, tobacco; however, the PEPC activity in the C3 plant merely increased by 2 to 3 times at most. There is another report by Matsuoka et al., Plant Physiol. 111: 949-957 (1996): For the purpose of studying the function of a promoter of the C4 photosynthesis gene, a fusion gene of the xcex2-glucuronidase (GUS) gene from E. coli and a 5xe2x80x2-flanking region (promoter region) of the PEPC gene was introduced into tobacco, whereby the GUS gene was expressed at a high level. There is also a report by Hudspeth et al., Plant Physiol. 98:458-464 (1992): As a C4 photosynthesis gene, the PEPC genome gene of maize containing the expression control region (promoter region) was introduced into tobacco; however, the PEPC activity merely increased by 2 to 3 times.
Thus, to the extent that the inventors are aware of, prior to the filing of Japanese Patent application No. 9-56742, on which the present application claims priority, there were no reports on examples where an enzyme involved in photosynthesis was expressed at a high level. Accordingly, there is a demand for techniques of expressing a photosynthesis gene of a C4 plant in a C3 plant at a high level, thereby enhancing the photosynthetic capacity of the C3 plant.
The present invention intends to overcome the above-mentioned problems, and its objective is to express a photosynthesis gene of a C4 plant in a C3 plant at a high level.
As described above, there is an example in which attempts have been made to introduce a PEPC genome gene involved in a photosynthetic pathway of a C4 plant, maize (poaceae) into a C3 plant, tobacco (solanaceae), resulting in low expression efficiency. The inventors of the present invention found that by introducing a gene containing (a) an expression control region of an enzyme involved in photosynthetic pathway of a C4 plant which is phylogenetically related to a C3 plant and (b) a structural gene for an enzyme involved in a photosynthetic pathway of a C4 plant which is phylogenetically related to a C3 plant, into the C3 plant, the expression efficiency of the enzyme involved in the C4 pathway of photosynthesis is remarkably enhanced, thereby achieving the present invention.
A C3 plant expressing a gene of a phylogenetically related C4 plant according to the present invention includes DNA containing (a) an expression control region of a gene for an enzyme involved in a photosynthetic pathway of a phylogenetically related C4 plant and (b) a structural gene for an enzyme involved in a photosynthetic pathway of the C4 plant, wherein the C3 plant expresses the enzyme encoded by the structural gene at a high level.
A method for producing a C3 plant which expresses a gene of a phylogenetically related C4 plant according to the present invention includes the steps of: transforming cells of the C3 plant with DNA containing (a) an expression control region of a gene for an enzyme involved in a photosynthetic pathway of a phylogenetically related C4 plant and (b) a structural gene for an enzyme involved in a photosynthetic pathway of the C4 plant; and regenerating the transformed cells of the C3 plant into the C3 plant; wherein the regenerated C3 plant expresses the enzyme encoded by the structural gene at a high level.
In one embodiment of the present invention, the C4 plant is a monocotyledonous plant, and the C3 plant is a monocotyledonous plant.
In another embodiment of the present invention, the C4 plant is a dicotyledonous plant, and the C3 plant is a dicotyledonous plant.
In another embodiment of the present invention, the DNA is a genome gene of the C4 plant.
In another embodiment of the present invention, the genome gene of the C4 plant is a genome gene of a C4 poaceous plant, and the C3 plant is a C3 poaceous plant.
In another embodiment of the present invention, the genome gene of the C4 poaceous plant is a genome gene for phosphoenolpyruvate carboxylase from maize, and the C3 poaceous plant is rice.
The present invention also relates to a C3 plant obtainable by a method according to the present invention, and a portion of the C3 plant.
In one embodiment of the present invention, the portion of a C3 plant according to the present invention is a vegetable.
In another embodiment of the present invention, the portion of a C3 plant according to the present invention is a fruit.
In another embodiment of the present invention, the portion of a C3 plant according to the present invention is a flower.
In another embodiment of the present invention, the portion of a C3 plant according to the present invention is a seed.
A C3 plant tissue expressing a gene of a phylogenetically related C4 plant according to the present invention includes DNA containing (a) an expression control region of a gene for an enzyme involved in a photosynthetic pathway of a phylogenetically related C4 plant and (b) a structural gene for an enzyme involved in a photosynthetic pathway of the C4 plant, wherein the C3 plant tissue expresses the enzyme encoded by the structural gene at a high level.
A method for producing a C3 plant tissue which expresses a gene of a phylogenetically related C4 plant according to the present invention includes the steps of: transforming cells of the C3 plant with DNA containing (a) an expression control region of a gene for an enzyme involved in a photosynthetic pathway of a phylogenetically related C4 plant and (b) a structural gene for an enzyme involved in a photosynthetic pathway of the C4 plant; and regenerating the transformed cells of the C3 plant into the C3 plant tissue; wherein the regenerated C3 plant tissue expresses the enzyme encoded by the structural gene at a high level.
In one embodiment of the present invention, the C4 plant is a monocotyledonous plant, and the C3 plant tissue is a tissue of a monocotyledonous plant.
In another embodiment of the present invention, the C4 plant is a dicotyledonous plant, and the C3 plant tissue is a tissue of a dicotyledonous plant.
In another embodiment of the present invention, the DNA is a genome gene of the C4 plant.
In another embodiment of the present invention, the genome gene of the C4 plant is a genome gene of a C4 poaceous plant, and the C3 plant tissue is a tissue of a C3 poaceous plant.
In another embodiment of the present invention, the genome gene of the C4 poaceous plant is a genome gene for phosphoenolpyruvate carboxylase from maize, and the C3 poaceous plant is rice.
A C3 plant seed expressing a gene of a phylogenetically related C4 plant according to the present invention includes DNA containing (a) an expression control region of a gene for an enzyme involved in a photosynthetic pathway of a phylogenetically related C4 plant and (b) a structural gene for an enzyme involved in a photosynthetic pathway of the C4 plant, wherein the C3 plant seed expresses, at least upon germination and growing, the enzyme encoded by the structural gene at a high level.
A method for producing a C3 plant seed which expresses a gene of a phylogenetically related C4 plant according to the present invention includes the steps of: transforming cells of the C3 plant with DNA containing (a) an expression control region of a gene for an enzyme involved in a photosynthetic pathway of a phylogenetically related C4 plant and (b) a structural gene for an enzyme involved in a photosynthetic pathway of the C4 plant; regenerating the transformed cells of the C3 plant into the C3 plant; and obtaining a seed from the C3 plant; wherein the C3 plant seed expresses, at least upon germination and growing, the enzyme encoded by the structural gene at a high level.
In one embodiment of the present invention, the C4 plant is a monocotyledonous plant, and the C3 plant seed is a seed of a monocotyledonous plant.
In another embodiment of the present invention, the C4 plant is a dicotyledonous plant, and the C3 plant seed is a seed of a dicotyledonous plant.
In another embodiment of the present invention, the DNA is a genome gene of the C4 plant.
In another embodiment of the present invention, the genome gene of the C4 plant is a genome gene of a C4 poaceous plant, and the C3 plant is a C3 poaceous plant.
In another embodiment of the present invention, the genome gene of the C4 poaceous plant is a genome gene for phosphoenolpyruvate carboxylase from maize, and the C3 poaceous plant is rice.
Thus, the invention described herein makes possible the advantages of (1) providing a C3 plant as well as a tissue and a seed thereof which express a C4 photosynthetic gene efficiently, and (2) further providing a technical foundation for enhancing the photosynthetic capacity of a C3 plant by conferring the C4 photosynthetic capacity to the C3 plant.
These and other advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description with reference to the accompanying figures.
FIG. 1 is a diagram showing a restriction enzyme map of a DNA fragment including the PEPC gene, where wider portions of the lines represent exons.
FIGS. 2A and 2B are diagrams showing a base sequence of about 8 Kb of a DNA fragment including the PEPC gene.
FIG. 3A and 3B are a continuation from FIG. 2B.
FIG. 4 is a continuation from FIG. 3B.
FIG. 5 is a diagram showing the binary vector pIG121-Hm.
FIG. 6 is a diagram showing a structure of the expression vector PEPCgenome/pBIH2.
FIG. 7 is a diagram showing PEPC activities of transgenic rice plants.